This research has as its overall goal the development of high performance liquid chromatography for use in biochemistry, especially in areas of amino acid, peptide and protein analysis. Work has focused on the development of stable and reproducible chemically bonded phases. Such phases are used in the separation of amino acids and peptides. New chemically bonded phases using wide pore small particle silica gel have also been developed. These phases permit separation based on (1) exclusion (2) hydrophobic or (3) ion exchange chromatography. By manipulation of the mobile phase good performance conditions for separation of a variety of proteins is achieved. Finally, the on-line coupling of liquid chromatography to mass spectrometry in the reversed phase mode has been developed. Here, a continuous microextractor after the reversed phase column permits desalting and the use of ion pair reagents for extraction and mass spectrometric analysis. This approach is now being examined for peptide separation and analysis.